Simple methods for synthesizing and purifying oligonucleotides are now in great demand due to the utility of synthetic oligonucleotides in a wide variety of molecular biological techniques. Automated synthesizers have proven useful in making targeted oligonucleotides. During oligonucleotide synthesis using an automated synthesizer, nucleotide monomers are progressively added to a nascent oligonucleotide chain. During each cycle, a percentage of the coupling reactions fail (i.e., no monomer addition occurs). Typically, coupling efficiency is 97-98% resulting in 2-3% of the reaction mixture being failed sequences. However, this error is multiplied as the oligonucleotide chain is lengthened. For example, in a typical 20-mer synthesis, the 20-mer product represents only 50-60% of the recovered oligonucleotide product. The remaining 40-50% represents failed sequences.
Since the chemical purity of a synthesized oligonucleotide is critical for any application, many techniques have been used to purify full length target nucleotides from truncated failed sequences. Among these are thin layer chromatography, polyacrylamide gel electrophoresis (PAGE), and high performance liquid chromatography (HPLC) by ion exchange using both normal and reversed phase modes. All of the aforementioned purification procedures can purify synthetic DNA only up to approximately 150 micrograms. In addition, all of these procedures require the reaction conditions to be varied if the phosphate backbone of the particular oligonucleotide has in any way been modified.
A method by which oligonucleotides having a desired sequence can be efficiently isolated in large quantities from a mixture of sequences, which includes failed sequences as well as sequences of interest, would be very useful.